DNA encoding proteins that inhibit Hsp70 function

ABSTRACT

Human heat-shock protein-binding proteins (HspBP-1 and HspBP-2) are disclosed with the polynucleotides which identify and encode them. Genetically engineered expression vectors and host cells comprising nucleic acid sequences encoding heat-shock protein-binding proteins (HspBP) are also disclosed, as well as methods for producing HspBP polypeptides and for detecting polynucleotide sequences that encode HspBP polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/109,351, filed by the same inventors on Nov. 20, 1998, and is acontinuation-in-part of U.S. patent application Ser. No. 09/444,336,filed on Nov. 19, 1999 now U.S. Pat. No. 6,410,713.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention generally relates to the field of molecular medicine andin particular to a novel set of heat-shock protein-binding proteins, andto polynucleotides encoding them, useful in the regulation ofphysiological events in which one or more 70 kiloDalton heat-shockproteins (Hsp70) are involved, such as normal development, cellularstress responses, heart disease, and cancer.

2. Description of the Related Art

Practically all organisms respond to heat by inducing the synthesis of agroup of proteins called the heat-shock proteins.

Although the details of this response vary among organisms, theinvolvement of Hsp70 and Hsp90gene families is known to be highlyconserved. More recently, it has come to be known that heat shockproteins can be induced by a variety of stress-related stimuli besidesheat: anoxia, ethanol and certain heavy metal ions also stimulateincreased expression and activity by these proteins. Hence, suchproteins commonly are more broadly referred to by those in the art asheat stress or, simply, stress proteins.

Interestingly, stress proteins also are present within cells undernon-stressful conditions (i.e. under normal physiological conditions).Genetic studies in bacteria and lower eukaryotes have demonstrated thatHsp70 is essential for growth at either high or normal temperatures,indicating a crucial role in normal cellular physiology. See generallyS. Lindquist and E. A. Craig, The Heat Shock Proteins, Annual Revue ofGenetics. 22:631-77 (1988).

Particular attention has been focused on Hsp70, a member of a multigenefamily whose genes are expressed under a wide variety of environmentalconditions and are found in all cells. As shown schematically in FIG. 1,Hsp70 and related proteins (such as Hsp72, Hsc70, and Grp78) contain anATPase domain, a substrate binding domain, and a coupling domain. S.Lindquist and E. A. Craig, Annual Revue of Genetics. 22:631-77 (1988).

In terms of function, studies have shown that Hsp70 plays a role in DNAreplication, transport of proteins across membranes, binding of proteinsto the endoplasmic reticulum, and uncoating clathrin coated vesicles. S.Lindquist and E. A. Craig, Annual Revue of Genetics. 22:631-77 (1988).Furthermore, Hsp70 is known to associate with nonsterified fatty acids,palmitic acid, stearic acid, and myristic acid and to be involved insignal transduction pathways in the cytoplasm. Hohfeld, Jorg, et al.,Hip, a Novel Cochaperone Involved in the Eukaryotic Hsc70/Hsp40 ReactionCycle. Cell vol. 83, 589-598 (Nov. 17, 1995).

Of these functions, perhaps the best studied has been the role of Hsp70as a “chaperone,” a protein that stabilizes other proteins againstaggregation and that mediates the folding of newly translatedpolypeptides in the cytosol and organelles. Proper functioning of Hsp70as a protein chaperone is dependent on its bound nucleotide state.Specifically, the ATP form of Hsp70 binds substrate very poorly andtherefore must be converted to the ADP form before the misfolded proteincan bind. Then, the high affinity of Hsp70 for ATP is utilized to“power” the protein folding and other functions of Hsp70, as much energyis generated by the hydrolysis of bound ATP.

The search for regulators of Hsp70 chaperone function has revealedregulatory factors that form complexes with Hsp70 and assist indetermining those substrates with which Hsp70 can associate. Forexample, the DNAJ-like proteins bind protein substrates exhibitingsecondary and tertiary structure but have very low affinity forpolypeptides in unfolded conformations. On the other hand, Hsp70proteins bind unfolded proteins best. Thus, by forming a complex withDNAJ-like protein, Hsp70 proteins can bind with many other proteins ofvarying conformation. Cyr, D. M. et al., DnaJ-like Proteins: MolecularChaperones and Specific Regulators of Hsp70. TIBS 19 (April 1994).

Other factors can regulate the substrate binding stability or ATPaseactivity of Hsp70. Hsp40 stimulates the ATPase of Hsp70 and thereforeresults in production of the ADP form of Hsp70, which facilitatesbinding to substrate. Another Hsp70 regulator, the Hip co-chaperoneprotein, binds to the ATPase domain of Hsp70, thereby promoting theassembly of chaperone complexes and prolonging the time window duringwhich a Hsp70 protein can interact stably with unfolded polypeptides.Hohfeld, Jorg, et al., Hip, a Novel Cochaperone Involved in theEukaryotic Hsc70/Hsp40 Reaction Cycle. Cell vol. 83, 589-598 (Nov. 17,1995). Similarly, a regulator named Hop modulates the binding of Hsp70to Hsp90, thereby stimulating Hsp70-mediated refolding of a denaturedprotein. Johnson, B. D., et al., Hop Modulates Hsp70/Hsp90 Interactionsin Protein Folding. JBC 273:6, pp. 3679-3686 (Feb. 6, 1998).

A potential regulator of Hsp70 is a 16-kDa protein that is a member ofthe Nm23/nucleoside diphosphate kinase family. This regulatormonomerized Hsc70 (a protein closely related to Hsp70) and assisted inreleasing Hsc70 from bound substrate. Leung, S. M. and L. E. Hightower,A 16-kDa Protein Functions as a New Regulatory Protein for Hsc70Molecular Chaperone and Is Identified as a Member of the Nm23/NucleosideDiphosphate Kinase Family. JBC 272:5, pp. 2607-2614 (Jan. 31, 1997).Also, the cysteine string protein, which is a secretory vesicle protein,and auxilin, a clathrin-associated protein, can both activate Hsc70ATPase activity. Chamberland, L. H. and R. D. Burgoyne, Activation ofthe ATPase activity of heat-shock proteins Hsc70/Hsp70 bycysteine-string protein. Biochem. J. 322, pp. 853-858 (1997); Braun, J.E. A., et al., The Cystein String Secretory Vesicle Protein ActivatesHsc70 ATPase. JBC 271:42, pp.25989-25993 (Oct. 18, 1996); Jiang, R. F.et al., Interaction of Auxilin with the Molecular Chaperone, Hsc70. JBC272:10, pp. 6141-6145 (Mar. 7, 1997).

Still other regulators of Hsp70 inhibit Hsp70-mediated refolding. TheRAP/HAP46 proteins, which inhibit binding of misfolded proteins toHsp70, and BAG-1, which causes the release of ADP from Hsp70, bothdown-regulate Hsp70 activity. Zeiner, M. et al., Mammalian proteinRAP46: an interaction partner and modulator of 70 dDa heat shockproteins. EMBO J. 16:18, pp. 5483-5490 (1997); Takayama, S. et al.,BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO J. 16:16,pp. 4887-4896 (1997).

Despite the fact that regulators of Hsp70 protein binding have beendiscovered and characterized, the functional regulation of Hsp70 is notyet understood. Moreover, the ability to directly abrogate or eliminateHsp70 ATPase activity through a selectively binding protein has notpreviously been known. Therefore, the discovery and isolation ofpolynucleotides encoding two isoforms of a human heat-shock proteinbinding protein (HspBP-1 and HspBP-2), is desirable because they providea means to investigate the effects of heat shock-protein regulation.Such regulation may have consequences in physiological pathways orconditions in which Hsp70 is known to be involved, such as development,apoptosis, cellular stress, heart disease, and cancer.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide the cloned polynucleotidesequences encoding novel human heat-shock protein-binding proteins.

A second object of the invention is to provide the deduced polypeptidesequences according to the cloned polynucleotide sequences encodingnovel human heat-shock protein-binding proteins.

Another object of the invention is to provide rat, mouse, and zebrafishgene homologues of novel human heat-shock protein-binding proteins.

Still another object of the invention is to provide a means forinhibiting the activity of Hsp70 and related proteins using novelheat-shock protein-binding proteins or fragments thereof.

Yet another object of the invention is to provide a means for inhibitionof the apoptotic activity of Hsp70 and related proteins using novelheat-shock protein-binding proteins or fragments thereof.

Still another object of the invention is to provide a means for rasingantibodies against novel heat-shock protein-binding proteins orfragments thereof.

In accordance with these objectives, the invention featuressubstantially purified human heat-shock protein-binding proteins(HspBP), designated HspBP-1 and HspBP-2, having the amino acid sequenceshown in SEQ ID NO:1 and in SEQ NO:2, respectively. Furthermore, theinvention features isolated and substantially purified polynucleotidesthat encode HspBP-1 or HspBP-2 having the nucleotide sequence shown inSEQ ID NO:3 and SEQ ID NO:4, respectively. Moreover, the inventionfeatures nucleic acid sequences encoding polypeptides, oligonucleotides,peptide nucleic acids, fragments, portions or antisense moleculesthereof, and expression vectors and host cells comprisingpolynucleotides that encode human HspBP and its mouse (HspBPM; SEQ IDNO:5), rat (HspBPR; SEQ ID NO:6), and zebrafish (HspBPF; SEQ ID NO:7)homologues. Finally, the invention features pharmaceutical compositionscomprising substantially purified HspBP.

Various other purposes and advantages of the invention will become clearfrom its description in the specification that follows and from thenovel features particularly pointed out in the appended claims.Therefore, to the accomplishment of the objectives described above, thisinvention consists of the features hereinafter illustrated in thedrawings, fully described in the detailed description of the preferredembodiments and particularly pointed out in the claims. However, suchdrawings and description disclose only some of the various ways in whichthe invention may be practiced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of Hsp70 showing its three functionaldomains.

FIGS. 2A and 2B shows the amino acid sequence alignments among humanHspBP-1 (SEQ ID NO:1) and HspBP-2 (SEQ ID NO:2) and the homologous mouse(HspBPM; SEQ ID NO:8),rat (HspBPR; SEQ ID NO:9), and zebrafish (HspBPF;SEQ ID NO:10) heat-shock protein binding proteins. Amino acid sequenceidentity among species is highlighted in black. Small stars (*) belowresidues indicate conservation of any amino acid among all species,while large stars (⋆) above residues indicate conservation of the aminoacid cysteine (C) among all species. When four of the five amino acidsfor a particular position are identical, a period (.) or a colon (:)below a residue indicate the degree to which chemical properties, suchas size and charge, are shared between the identical and non-identicalresidues, with a period indicating partial chemical relatedness and acolon indicating high chemical relatedness. The dashes (-) indicate thata protein lacks an amino acid at that particular position of thealignment.

FIG. 3 shows a Western blot analysis of HspBP1. Proteins from a lungadenocarcinoma cell line (lane 1) and transcribed/translated HspBP1(lane 2) and were separated on SDS polyacrylamide gels and transferredto nitrocellulose paper. Lane 1 was then incubated with anti-HspBP1antibody and detection of antibody binding was performed usingSuperSignal ULTRA (Pierce). Lane 2 was exposed to film to detect ³⁵Slabeled proteins from the coupled transcription/translation. Numbers onthe left side are molecular weight markers in kDa.

FIG. 4 shows the binding of HspBP1 to the ATP-binding domain of Hsp70.The ATP-binding fragment of Hsp70 (amino acids 1-351) was incubated withor without the His-tagged HspBP1. These solutions were then incubatedwith a Ni²⁺affinity resin which binds the His-tagged HspBP1 and anyassociated proteins. The resin was centrifuged and washed several timeswith wash buffer. The bound proteins were eluted with 1M imidazolebuffer and analyzed by Western blotting with both anti-6XHis antibodyand anti-Hsp70 antibody. Hsp70 (1-351) bound to the resin only in thepresence of HspBP1 (lane 3). Lane 1, HspBP1 alone on resin; lane 2,Hsp70 (1-351) alone on resin; lane 3, HspBP1 and Hsp70 (1-351); lane 4,HspBP1 standard; lane 5, Hsp70 (1-351) standard.

FIG. 5 shows HspBP1 binding to Hsp70 in a tissue homogenate. His-taggedHspBP1 was first bound to Ni²⁺affinity resin. A crude homogenate ofbovine heart was prepared and incubated with the resin for 15 min. at30° C. The resin was centrifuged and the supernates were saved. Theresin was then rinsed several times with buffer and proteins were elutedby incubation with 1M imidazole buffer. The eluted proteins wereanalyzed by separation on polyacrylamide gels containing SDS and stainedwith Coomassie blue (Panel A) or Western blotted and probed with bothanti-Hsp70 antibody and anti-6XHis antibody (Panel B). Lane 1 containsproteins eluted from resin without HspBP1 bound and Lane 2 containsproteins eluted from resin with HspBP1 bound.

FIG. 6 shows a Northern blot of poly A⁺ RNAs from human tissues. ANorthern blot (Clontech Lab., Inc.) containing poly A⁺ RNA from humanheart (1), brain (2), placenta (3), lung (4), liver (5), skeletal muscle(6), kidney (7) and pancreas (8) was probed with either human HspBP1cDNA (panel A), Hsp70 cDNA (panel B) or Hsp40 cDNA (panel C). Numbers onthe left are molecular weight markers in kilobases.

FIG. 7 shows the effect of HspBP1 on Hsp70 ATPase activity. ATPaseactivity was determined using 1.4 μM Hsp70, 7.9 μM HspBP1 and 16.5 nMHsp40. Effects of HspBP1 on Hsp70 ATPase activity (A) and Hsp40stimulated activity (B) were determined as described in the ExperimentalProcedures section of the Detailed Description. The blank control,endogenous Hsp70, and HspPB1 activities were subtracted to determine theeffect on the Hsp40 stimulated Hsp70 activity (C). ●, Hsp70+Hsp40; ♦Hsp70+HspBP1; ◯, Hsp70+Hsp40+HspBP1; ▪, Hsp70 alone; ▴, HspBP1 alone; □,blank.

FIG. 8 shows the inhibition of nucleotide binding by HspBP1. Sampleswere prepared as described for the ATPase assays using 20 μM [α-³⁵S]ATPand 12 μM HspBP1. Samples were removed (10 μl) after 10 min. ofincubation and unbound nucleotides were removed using spin columns.Bound nucleotides were processed as described above to separate ATP fromADP. A, total nucleotide bound; B, ATP bound; C, ADP bound.

FIG. 9 shows luciferase renaturation inhibition by HspBP1. Luciferasewas denatured by heating and then placed on ice. The denatured enzymewas added to either rabbit reticulocyte lysate (A) or a defined systemcontaining Hsp70, Hsp40 and Hsp90 (B) in the absence or presence ofincreasing amounts of HspBP1. Aliquots were removed and assayed foractivity. Activities are compared to luciferase renatured withoutHspBP1. Points are averages of triplicate assays and standard deviationswere less than 5% of the mean.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by those of ordinary skillin art of the invention. For example, see the definitions provided byU.S. Pat. No 5,955,312 by Hillman and Goli, which is incorporated hereinby reference. All publications mentioned herein are incorporated byreference for the purpose of describing and disclosing the cell lines,vectors, and methodologies which might be used in connection with theinvention.

The invention is based on the discovery of novel polynucleotidesencoding two isoforms of human HspBP, and the use of thesepolynucleotides and proteins in discovering and isolating the homologouspolynucleotides and proteins of several different species, includingmouse, rat, and zebrafish.

The polynucleotides and proteins of the invention are useful forresearch on pathways in which active Hsp70 and related proteinsparticipate, such as apoptosis, development, and signal transduction.Furthermore, the invention is useful in the research and in thetreatment of maladies involving active Hsp70, such as various types ofcancer and heart disease.

For example, it is known that hypoxic stress is a signal that increasesthe amount of Hsp70 in cardiac tissue, whereupon Hsp70 helps cellssurvive by binding to partially denatured proteins and assisting in therefolding of these proteins into more stable native structures. Suchassistance would be extremely important in proving protection to theheart during periods of hypoxia such as during an infarct or duringsurgery when blood flow to the heart may be temporarily halted. Thus,discovering, characterizing, and devising ways to down-regulate theexpression or activity of Hsp70-inhibiting proteins, such as through aHspBP, is clearly useful.

It is also known that harmful conditions, including oxidative stress andUV radiation, can cause programmed cell death (apoptosis). Hsp72, amember of the Hsp70 family, has been shown to inhibit a signaltransduction pathway leading to programmed cell death by preventingstress-induced activation of Jun N-terminal kinase, JNK. Gabai, V. L. etal. Hsp70 Prevents Activation of Stress Kinase. JBC 272:29, pp.18033-18037 (Jul. 18, 1997). Moreover, Hsp70 is known to block theapoptotic process by blocking the activation of the caspase proteasecascade. Mosser, D. D., et al., Role of the Human Heat Shock ProteinHsp70 in Protection Against Stress-Induced Apoptosis. Mol. and Cell.Biol., 17:9, pp. 5317-5327 (September, 1997). Thus, HspBP may play arole in promoting apoptosis by halting the inhibitory action of Hsp72 onJNK. By promoting apoptosis, HspBP may be useful in the killing of, forexample, cancer cells.

Although many different methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods, devices, and material are nowdescribed.

The invention encompasses polypeptides comprising the amino acidsequences of SEQ ID NO:1 (HspBP-1; GenBank Acession Number AF093420) andSEQ ID NO:2 (HspBP-2; GenBank Acession Number AF187859). HspBP-1 is 359amino acids long, while HspBP-2 is 362 amino acids in length. Thisdifference in length is accounted for by the presence of 3 additionalglycine residues in HspBP-2 at a glycine-rich region beginning atresidue 25.

While full length HspBP polypeptides are used in the experimentsdescribed herein, it should be recognized by all those skilled in theart that fragments of the full length polypeptides are also within thescope of the invention. For example, immunologically-active fragments ofHspBP-1 are recognized by rabbit serum antibodies as described below.Moreover, fragments of HspBP polypeptides are thought to be involved inbinding to the ATP-binding domain of Hsp70 and to regulate Hsp70 ATPaseactivity.

Therefore, the invention also relates to peptide fragments of HspBPhaving at least one biological activity, including, but not limited to,immunological activity (binding to antibody), Hsp70 ATPase activityregulation, or Hsp70 binding.

The invention also encompasses polynucleotides which encode HspBP. Thus,any nucleic acid sequence which encodes an amino acid sequence of aHspBP can be used to produce recombinant molecules which express HspBp.In a particular embodiment, the invention comprises the polynucleotidesequences encoding HspBP-1 (SEQ ID NO:3) and HspBP-2 (SEQ ID NO:4).Polynucleotide fragments encoding portions of HspBP's are also part ofthe invention. For example, polynucleotide fragments of HspBP capable ofhybridization with polynucleotide sequences of HspBP DNA or RNA as aprobe or sense/antisense complex, are included in the scope of theinvention.

Furthermore, methods of detecting a target polynucleotide sequence in asample, such as, SEQ ID NO. 3, SEQ ID NO. 4, or fragments thereof, areincluded. For example, a method of detecting may include hybridizing asample with a probe comprising at least 20 contiguous nucleotides,wherein the probe includes a sequence complementary to the targetpolynucleotide in the sample, and which probe specifically hybridizes tothe target polynucleotide, under conditions whereby a hybridizationcomplex is formed between the probe and the target polynulceotide orfragments thereof. Then, the presence or absence of said hybridizationcomplex is detected.

The invention further encompasses HspBP variants. A preferred variant isone having at least 90% amino acid sequence similarity to the HspBPamino acid sequences identified by SEQ ID NO:1 and SEQ ID NO:2. Mostpreferably, however, is a HspBP variant having at least 95% amino acidsequence similarity to SEQ ID NO:1 or SEQ ID NO:2.

As known by those skilled in the art, many commonly available computerprograms can be used to search for sequence variants. For example, boththe nucleotide and derived amino acid sequences of the human HspBP wereused to search GenBank™, and no matches to known genes or proteins werefound. However, when searching the GenBank™ EST data base with theprograms BLAST and tBLASTn (National Center for BiotechnologyInformation), a number of significant matches were found in human,mouse, and rat sequences. None of these sequences were for knownproteins.

The deduced amino acid sequences of the HspBP of humans (HspBP-1; SEQ IDNO:1 and HspBP-2; SEQ ID NO:2), mice (HspBPM; SEQ ID NO:8), rats(HspBPR; SEQ ID NO:9), and zebrafish (HspBPF; SEQ ID NO:10) are shown inFIGS. 2A and 2B in alignment using the CLUSTALW computer program. Asindicated by the black shading in FIGS. 2A and 2B, the amino acidsequences of HspBP for all species tested are highly conserved.

It will be appreciated by those skilled in the art that, as a result ofthe degeneracy of the genetic code, a multitude of HspBP-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.The invention contemplates every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequenceencoding naturally occurring HspBP, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode HspBP and their variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring transcription sequences under appropriately selectedconditions of stringency, it can be advantageous to produce nucleotidesequences encoding HspBP or their derivatives possessing a substantiallydifferent codon usage. For example, codons may be selected to increasethe rate at which expression of the peptide occurs in a particularprokaryotic or eukaryotic expression host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encodingHspBP and their derivatives without altering the encoded amino acidsequences include the production of RNA transcripts having moredesirable properties, such as a greater stability or half-life, thantranscripts produced from the naturally occurring sequence.

As known by one skilled in the art, a DNA sequence, or portions thereof,encoding HspBP and their derivatives may be produced entirely bysynthetic chemistry. Subsequently, the synthetic nucleotide sequence maybe inserted into any of the many available DNA vectors and cell systemsusing reagents that are commonly available. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingHspBP or any portion thereof.

Also included within the scope of the invention are polynucleotidesequences that are capable of hybridizing to the nucleotide sequences ofSEQ ID NO:3 or SEQ ID NO:4 under various conditions of stringency.Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex or probe, as taught in Berger andKimmel (1987, Guide to Molecular Cloning Techniques, Methods inEnzymology, v.152, Academic Press, San Diego, Calif.).

Natural, modified, or recombinant nucleic acid sequences may be ligatedto a heterologous sequence to encode a fusion protein. One may, forexample, screen a peptide library for inhibitors of HspBP activity byencoding a chimeric HspBP that can be detected by a commerciallyavailable antibody. In addition, a fusion protein may be engineered tocontain a cleavage site located between the HspBP encoding sequence andthe heterologous protein sequence, so that HspBP may be cleaved andpurified away from the heterologous moiety.

Methods well known in the art can be used to construct expressionvectors containing sequences encoding HspBP and appropriatetranscriptional and translational control elements. Methods may includein vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination in a variety of expression vector/host systems,such as bacteria transformed with recombinant bacteriophage or plasmidsor insect cell systems infected with viral expression vectors such asthe baculovirus. These methods are described in standard laboratoryreferences, such as Sambrook, J. et al. Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. (1989).

Altered nucleic acids encoding HspBP which may be used in accordancewith the invention include deletions, insertions or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent HspBP. The protein may also showdeletions, insertions or substitutions of amino acid residues whichproduce a silent change and result in functionally equivalent HspBP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of HspBP is retained. For example, negativelycharged amino acids aspartic acid and glutamic acid might be substitutedfor one another.

Also included within the scope of the invention are alleles encodingHspBP. As used herein, an “allele” or “allelic sequence” is analternative form of the nucleic acid sequence encoding HspBP. Allelesresult from a mutation, i.e. a change in the nucleic acid sequence, andgenerally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one ormany allelic forms. Common mutational changes which give rise to naturaldeletions, additions or substitutions of amino acids. Each of thesetypes of changes may occur alone, or in combination with the others, oneor more times in a given sequence.

Many ways exist in the art by which HspBP may be used therapeutically.Examples include, but are not limited to, administering HspBP throughthe introduction of an expression vector into a subject for in vivotherapy, administering a vector expressing antisense of a polynucleotideencoding HspBP, or administering HspBP as part of a pharmaceuticalcomposition. Depending on the route of administration, appropriateagents for use in combination with HspBP for therapy may include anyconventional pharmaceutical carrier such as saline or buffered saline(intravenous dosing) and dextrose or water (oral dosing). Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

In order to further illustrate the invention, the following example isprovided. However, this example is not intended in any way to limit theinvention.

EXAMPLE Experimental Procedures

Screening of Yeast Library and Cell Cultures.

The two-hybrid system was used to screen for Hsp70 interacting proteins.A portion of the cDNA for human Hsp70 (kindly provided by R. Morimoto,Northwestern University) coding for amino acids 1-351 was inserted intothe yeast vector pAS2 (Clontech Laboratories, Palo Alto, Calif.) andused as the “bait plasmid” for the two-hybrid screening procedures. Theyeast transfected with this plasmid expressed human Hsp70 as determinedby Western blot analysis. These yeast would not grow on plates lackinghistidine and were negative for β-galactosidase activity indicating thatHsp70 alone cannot activate the reporter genes and therefore will notresult in false positives.

A human heart CDNA library (Clontech Laboratories, Palo Alto, Calif.)containing 3×10⁶ independent clones was screened. This library was madein the pGAD10 cloning vector which had cDNAs fused to the activationdomain (AD) of the GAL4 transcription activator. Methods for screeningof the library were according to the manual provided by Clontech. Clonesthat lacked the DNA-BD/target plasmid but retained the AD/libraryplasmid were isolated using cycloheximide selection (MatchmakerSupplement Kit, Clontech). The candidate Leu⁺, Trp⁻ clones were thenmated to Y187 (MATα) yeast strains carrying different test plasmids.Diploids from the mating were selected (Trp⁺, Leu⁺, His⁺) and screenedfor the ability to produce β-galactosidase.

Lung adenocarcinoma cells (#3263) were kindly provided by the Universityof Arizona Cancer Center Cell Culture Core Facility.

A cell pellet (approximately 100 μl) was frozen and thawed, suspended inan equal volume of 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 2mMPMSF, centrifuged in a microfuge and the supernate was used for furtheranalysis.

DNA Sequencing and Northern Blot Analysis.

Plasmids were sequenced in both directions by the Laboratory ofMolecular Systematics and Evolution at the University of Arizona.Oligonucleotides for sequencing were purchased from GenosysBiotechnologies (The Woodlands, Tex.). A Northern blot of human tissuemRNAs was purchased from Clontech Laboratories (Palo Alto, Calif.) andprobed according to the procedure supplied by Clontech Laboratoriesusing ExpressHyb hybridization solution. HspBP1 from nucleotide457-1000, Hsp70 from nucleotide 841-1765 and Hsp40 from nucleotides582-1043 were labeled and used as probes.

Bacterial Expression and Transcription/Translation.

The cDNA for the ATP-binding domain of Hsp70 (amino acids 1-351) wasinserted into the expression vector pET28a (Novagen, Madison, Wis.)minus the His-tag and expressed in bacteria. The protein was purified bysolubilizing inclusion bodies in 6M guanidine HCl in binding buffer (0.5M NaCl, 5 mM imidazole and 20 mM Tris-HCl, pH 7.9) followed by dialysisagainst binding buffer.

The CDNA for HspBP1 was inserted into pET28a and expressed as a fusionprotein containing the His-tag. Mutagensis by PCR was performed in pAS2to create a NdeI site at the translation initiation region. Themutagenized fragment was subcloned into the TA vector (Invitrogen,Carlsbad, Calif.). Next, the original HspBP1 insert was removed frompAS2 by cutting with EcoRI and subcloning into the EcoRI site of pET28a.This was cut with NdeI and KpnI and this region was removed and replacedby the mutated fragment removed by digestion with NdeI and KpnI from theTA vector. The protein was purified over a His Bind affinity resin(Novagen, Madison, Wis.) following the manufacture's procedure with theaddition of 0.5% NP40 to all buffers except the elution buffer.

To insert HspBP1 cDNA into the proper vector for coupledtranscription/translation, the cDNA for HspBP1 was subcloned into pET5ausing the Nde I and EcoR1 sites and coupled transcription/translationwas done using the TNT T7 Quick Coupled Transcription/Translation System(Promega, Madison, Wis.) and [³⁵S]methionine (Amersham, ArlingtonHeights, Ill.)

Antibody Production and Western Blot Analysis.

HspBP1 was prepared as described above and further purified byseparation on a SDS polyacrylamide gel and the band was removed byelectroelution. This preparation was used for antibody production.Antibodies were produced in rabbits by Animal Pharm Services, Inc.(Healdsburg, Calif.). Specific antibodies were purified from serum byinitial affinity purification of IgG using a protein A column followedby an HspBP1 affinity column to purify specific antibodies.

SDS sample buffer was added to the lung adenocarcinoma cell lysate andthe transcription/translation product. All samples were then heated at95° C. for 5 min., centrifuged and the supernate was analyzed on a 12.5%SDS gel and transferred to nitrocellulose paper. The blot was blocked byincubation with Tris-saline (154 mM NaCl, 10 mM Tris, pH 7.5) containing5% nonfat milk. Blots were then incubated with anti-HspBP1 antibody orpreimmune IgG (0.1 μg/ml) overnight. Detection of antibody binding wasperformed using SuperSignal ULTRA (Pierce Chemical Co., Rockford, Ill.).

Hsp70 Binding to HspBP1.

The ATP-binding domain of Hsp70 (amino acids 1-351) was inserted intothe expression vector pET28a (minus the His-tag), expressed in bacteriaand purified by isolation of inclusion bodies. The truncated Hsp70 wasincubated with or without the His-tagged HspBP1. These solutions werethen incubated with a Ni²⁺affinity resin which binds the His-taggedHspBP1 and any associated proteins. HspBP1 alone or Hsp70 (1-351) alonewere also incubated with the resin. The resin was centrifuged and washedseveral times with wash buffer. The bound proteins were eluted with 1 Mimidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9) and analyzed by Westernblotting with both anti-6×His antibody (Clontech Laboratories, Inc.,Palo Alto, Calif.) and anti-Hsp70 antibody (StressGen, Victoria, BC).

Binding to proteins in a total homogenate was done by first bindingHis-tagged HspBP1 to the Ni²⁺affinity resin. A crude homogenate ofbovine heart was prepared and incubated with the resin for 15 min. at30° C. The resin was centrifuged and the pellets were saved. The resinwas then rinsed several times with wash buffer and proteins were elutedby incubation with 1M imidazole buffer as above. The eluted proteinswere analyzed by separation on polyacrylamide gels containing SDS andstained with Coomassie blue or Western blotted and probed with bothanti-Hsp70 antibody and anti-6×His antibody.

ATPase Assays and ATP Binding.

ATPase assays were performed in 20 μM [α-³⁵S]ATP (0.631 mCi/μM), 4 mMHepes, pH 7.4, 7.5 mM KCl, 0.45 mM MgAcetate and 80 μM DTT. Humanrecombinant Hsp70 (StressGen, Victoria, BC), HspBP1 and Hsp40 (kindlyprovided by Dr. E. Vierling, University of Arizona) were added at theindicated amounts. Assays were incubated at 37° C. and aliquots (1 μl)were removed at 0, 5, 10 and 15 min., spotted on PEI cellulose paper (J.T. Baker, Inc., Phillipsburg, N.J.) and developed in 1 M formic acid and0.5 M LiCl to separate ATP and ADP. Radioactivity was quantitated on aPackard Instant Imager (Meriden, Conn.). ATP binding was determinedafter 10 min., samples were removed (10 μl) and unbound nucleotides wereremoved using spin columns (ProbeQuant G-50, Pharmacia Biotech,Piscataway, N.J.). Bound nucleotides were processed as described aboveto separate ATP from ADP.

Renaturation of Luciferase.

Assays to measure the renaturation of luciferase in rabbit reticulocytewere done following the procedure of Schumacher et al. (1996)Biochemistry 35, 14889-14898. Luciferase (Sigma Chemical Co., St. Louis,Mo.) at 100 nM was dissolved in 25 mM tricine, pH 7.8, 8 mM Mg SO₄, 0.1mM EDTA, 10 mg/ml bovine serum albumin, 10% glycerol, and 0.25% tritonX-100. The protein was denatured by heating at 40° C. for 15 min. andthen placed on ice. The enzyme was then diluted 10-fold by addition torabbit reticulocyte lysate (Green Hectares, Oreg., Wis.) containing anATP regeneration system and incubated at 25° C. for 90 min. Samples (5μl) were removed and assayed for luciferase activity by addition to 120μl of 25 mM tricine, pH 7.8, 8 mM MgCl₂, 0.1 mM EDTA, 12 mM DTT, 100 μMD-luciferin, 240 μM coenzyme A, and 0.5 mM ATP. Light production wasmeasured in a Turner luminometer for 15 seconds.

Renaturation of recombinant firefly luciferase (Promega, Madison, Wis.)in a defined system was performed following the procedure of Johnson etal. (1998) J. Biol. Chem. 273, 3679-3686. Luciferase was diluted to 100nM into above buffer and denatured by heating at 40° C. for 8 min andthen placed on ice. The enzyme was then diluted 10-fold into 10 mM Tris,pH 7.5, 3 mM MgCl₂, 50 mM KCl and 2 mM dithiothreitol containing 800 nMhuman recombinant hsp70, 140 nM human recombinant Hsp90 alpha(StressGen, Victoria, BC), 160 nM Hsp40 and an ATP regenerating systemand incubated at 25° C. for 4 hours. Samples were assayed as above.

Computer Analysis.

Database searches were done using the BLAST server at the NationalCenter for Biotechnology Information and the programs BLAST and tBLASTn.Isoelectric point and molecular weight determinations were done usingthe ExPASy molecular biology server at the Geneva University Hospitaland University of Geneva, Geneva, Switzerland.

Results and Discussion

Isolation of cDNAs Using the Two-Hybrid System.

A human heart CDNA library was screened and three colonies were isolatedthat grew on the trp⁻, leu⁻, his⁻ (triple minus) plates and werepositive for β-galactosidase activity. Yeast containing the isolatedlibrary plasmids were mated to yeast containing the cDNA for thetruncated Hsp70 and once again these colonies grew on the triple minusplates and were positive for β-galactosidase activity. Mating of yeastcontaining the library plasmids with other controls (plasmid with noinsert, plasmid with an insert other than Hsp70) did not producecolonies when grown on triple minus plates indicating that theinteraction is specific for Hsp70 and the library plasmids.

The cDNA insert sizes were 1.5, 1.6 and 1.6 kilobases. Nucleotidesequencing revealed open reading frames that code for proteins ofapproximately 40 kDa. Two of the clones were identical (HspBP2) and thethird (HspBP1) differed in the coding region by having codons for 6consecutive glycines whereas HspBP2 had codons for 9 glycines. HspBP2also had two polyadenylation signals whereas HspBP1 had only the firstpolyadenylation signal. All results reported herein were done withHspBP1 (Seq. Listing No. 1). The sequence 5′ to the initiation codon didnot contain a termination codon therefore it was possible the codingregion was not complete. Polymerase chain reaction (PCR) was used toamplify products from the plasmid library using a primer for a sequence3′ to the codons for the glycines and a primer in the flanking pGAD10vectors. These experiments extended the sequence 53 bases to the 5′-end(underlined sequence in FIG. 1). This sequence lacked initiation codonsin any reading frame but did contain a stop codon in the same readingframe as the open reading frame for the protein. Therefore, the firstATG after the stop codon has been assigned as the initiation codon. Thecalculated pI for the protein is 5.13 and the calculated molecularweight is 39,302. Both the nucleotide and derived amino acid sequenceswere used to search GenBank and no matches to known genes or proteinswere found. However, when searching the GenBank EST database with theprograms blast and tblastn, a number of significant matches were foundin human, mouse and rat sequences. None of these sequences were forknown proteins. The protein sequence was also analyzed for domaincharacteristics using a number of different programs and no similaritieswere found.

A polyclonal antibody against HspBP1 was prepared and used to detectHspBP1 in cell homogenates by western blots (FIG. 3). The cDNA forHspBP1 was transcribed and translated in vitro and this protein productwas compared to the protein detected in cells. The recombinant HspBP1has a slightly higher molecular weight due to additional amino acidsincluding the His tag (not shown). The in vitro transcribed andtranslated product (lane 2) is the same size as the protein found in thecells (lane 1), confirming that the cDNA contains the entire proteincoding region.

A fragment of Hsp70 (amino acids 1-351) identical to the ATPase domainthat was used in screening the yeast library was expressed in bacteriaand purified. The fragment bound to a Ni²⁺ affinity column in thepresence of His-tagged HspBP1 (FIG. 4, lane 3) providing furtherevidence for an interaction between HspBP1 and the ATPase domain ofHsp70. The fragment was not retained on the column in the absence ofHspBP1 (FIG. 4, lane 2). In a second set of experiments, His-taggedHspBP1 was first bound to the Ni²⁺ affinity resin and binding toproteins in a homogenate of bovine heart was determined. Westernblotting revealed a strong immunoreactive band for Hsp70 that was elutedfrom the resin when HspBP1 was first bound (FIG. 5, panel B, lane 2).This band was absent if HspBP1 was not bound to the resin first (FIG. 5,panel B, lane 1). Further analysis on a lower percentage gel resolvedthe Hsp70 into two bands (not shown), which is consistent with bothHsp70 and Hsc70 binding to HspBP1. An additional immunoreactive bandlarger than Hsp70 is eluted in the presence or absence of HspBP1. Thisprotein binds to the resin nonspecifically and reacts with theanti-Hsp70 antibody and could not be detected by protein staining (panelA). Protein staining revealed both the eluted HspBP1 (Panel A, lowerarrow) and Hsp70 (upper arrow). Additional bands are present above Hsp70and below HspBP1 and are eluted in the presence or absence of HspBP1.

A Northern blot of human tissues (FIG. 6) was probed with a region ofHspBP1 cDNA (panel A), Hsp70 cDNA (panel B) and Hsp40 cDNA (panel C).Heart and skeletal muscle contain the highest amounts of HspBP1 mRNA.These two tissues also exhibit relatively high levels of Hsp70 and Hsp40mRNAs. However, there is not a consistent correlation between the amountof HspBP1, Hsp70 and Hsp40 mRNAs in the other tissues. These resultssuggest that there is a tissue specific expression of HspBP1 and thisexpression is not dependent on the amount of Hsp70 or Hsp40 mRNA. In alltissues the size of the HspBP1 mRNA is approximately 1.7 kb.

The next series of experiments were conducted to determine the effect ofHspBP1 on Hsp70 enzymatic activity. HspBP1 had a slight stimulatoryeffect on the Hsp70 ATPase activity in the absence of Hsp40 (FIG. 7,panel A) but this is due to the low ATPase activity found in the HspBP1preparations (FIG. 7, panel B). This is most likely a contaminant thatvaries in different preparations. The Hsp70 ATPase could be stimulatedby Hsp40 (FIG. 7, panels A,B) and this increase in activity wasinhibited by HspBP1 (FIG. 7, panel B). The ATPase activities for Hsp70and HspBP1 alone were subtracted from the activity of Hsp70+Hsp40+HspBP1to determine the effect on the Hsp40 stimulated activity (FIG. 7, panelC). These data clearly indicate that HspBP1 inhibits the Hsp40stimulation of Hsp70 activity.

The effect of HspBP1 on nucleotide binding to Hsp70 under steady-stateconditions was next examined to explore the mechanism by which HspBP1inhibits Hsp70 ATPase. In the absence of Hsp40, the majority ofnucleotide bound to Hsp70 is in the form of ATP which is consistent withan unstimulated ATPase activity (FIG. 8). HspBP1 inhibited nucleotidebinding by approximately 30% and this was due to a decrease in ATPbinding. In the presence of Hsp40, the total amount of nucleotidebinding doubles and the ratio of ADP to ATP increases, consistent with astimulation of ATPase activity, the production of ADP and ATP turnover.In the presence of HspBP1 and Hsp40 the amount of total nucleotide bounddecreased to half the amount and this is due to a decrease in both ATPand ADP bound. These results are consistent with a decrease in ATPbinding which would decrease the amount of nucleotide available forhydrolysis and therefore result in a decrease in ADP bound. We concludefrom these experiments that the inhibition in Hsp70 ATPase activity byHspBP1 is due to a decrease in ATP binding.

The ADP form of Hsp70 binds substrate whereas the ATP form cannot,therefore a decrease in ATP binding and an inhibition of Hsp40stimulated ATPase suggests that HspBP1 could inhibit the ability ofHsp70 to renature a substrate. Reticulocyte lysate contains thenecessary components including Hsp70 for renaturation of denaturedfirefly luciferase. The effect of HspBP1 on Hsp70 -dependent proteinrenaturation was analyzed using this system. Experiments were done withvarying concentrations of HspBP1 and measuring the amount of luciferaserenatured after 90 min. (FIG. 9, panel A). HspBP1 inhibited renaturationof luciferase with a half-maximal inhibition at 2 μM. The reticulocytelysate is an undefined system containing many unknown proteins,therefore, similar refolding experiments were done in a defined systemas described by Johnson et al. (1998) J. Biol. Chem. 273, 3679-3686. Inthe defined system HspBP1 again inhibited renaturation of luciferase isa dose-dependent manner 20 with a similar half-maximal inhibitionconcentration (FIG. 8, panel B). Maximum inhibition was approximately50% whereas in the recticulocyte lysate inhibition was over 90%. Theseexperiments were done with different sources of luciferase and this mayexplain the difference. These data indicate a relatively tightinteraction of HspBP1 with Hsp70. As a comparison, the dissociationconstant for auxilin (an activator of Hsc70 ATPase activity) with Hsc70was determined to be 0.6 μM (13).

Recently, numerous proteins have been identified as regulators of Hsp70mediated renaturation of misfolded proteins indicating that this is acomplex system that is far from clearly defined. The findings reportedin this paper increase this complexity by the addition of anotherprotein (HspBP1) that may have potential in vivo regulatory propertiesof Hsp70. Proper functioning of Hsp70 as a protein chaperone isdependent on its bound nucleotide state. The ATP form of the proteinbinds substrate very poorly and therefore must be converted to the ADPform before the misfolded protein can bind. This conversion is catalyzedby another heat stress protein named Hsp40. As reported here, HspBP1inhibits the Hsp40 stimulated Hsp70 ATPase by inhibiting ATP bindingtherefore blocking the production of ADP. The end result is theinhibition of Hsp70 mediated refolding as seen in the HspBP1 inhibitionof renaturation of denatured luciferase in the rabbit reticulocytelysate and in a defined system. Other regulators of Hsp70 activityaffect ADP binding. For example, Hip stimulates the renaturation of adenatured substrate by decreasing the release of ADP, whereas BAG-1inhibits Hsp70 activity by increasing the release of ADP.

The Northern blot analysis indicates that heart and skeletal musclecontain the highest amounts of HspBP1 mRNA. It is not known if thisreflects the relative amounts of the protein in these tissues. A highamount in heart muscle does pose some interesting speculation as to thefunction in this tissue. Recent studies in Xenopus have reported thatthe heart is the most thermal sensitive of the organs examined withrespect to activation of heat shock transcription factor (HSF) bindingand an increase in Hsp70 mRNA and protein levels. HspBP1 may bind to theendogenous Hsp70 rendering it inactive and thereby cause a feedbackmechanism whereby the cell senses a lower amount of active Hsp70 andresults in an increased Hsp70 expression. Through this mechanism theheart would have lower levels of active Hsp70 and therefore a loweramount of stress would be required to activate synthesis of more Hsp70.

The experiments reported here indicate that HspBP1 may regulate Hsp70mediated refolding of denatured proteins, however, it remains to be seenif this is the in vivo function. It is possible that HspBP1 may beinvolved in other Hsp70 regulated activities such as apoptosis via thestress activated protein kinase pathway (SAPK). For instance, cell deathcaused by tumor necrosis factor (TNF) can be prevented by overproductionof Hsp70 (24). Recently, others have provided evidence that Hsp70 caninhibit the activation of Jun N-terminal kinase (JNK) and therebyinhibit phosphorylation of c-JUN by JNK resulting in an inhibition ofapoptosis via this pathway. Further down this pathway, Hsp70 has beenshown to inhibit the processing of caspase-3 from the inactivepro-caspase and thereby inhibiting the proteolytic activity of thisenzyme and cell death. These, then, are potential parts of the pathwaythat may be regulated by HspBP1.

While this example is contemplated to be the preferred mode, it will beunderstood by those in the art that numerous alternative methodologiesmay be successfully practiced in lieu of the preferred method describedherein.

1. An isolated polynucleotide encoding the amino acid sequence of SEQ IDNO:1.
 2. An isolated polynucleotide of claim 1, wherein saidpolynucleotide comprises SEQ ID NO.3.
 3. A hybridization probecomprising a polynucleotide of claim 1 and a detectable label.
 4. Ahybridization probe comprising the polynucleotide of claim 2 and adetectable label.
 5. A polynucleotide which is fully complementary tothe polynucleotide of claim
 1. 6. A polynucleotide which is fullycomplementary to the polynucleotide of claim
 2. 7. A hybridization probecomprising the polynucleotide of claim 5 and a detectable label.
 8. Ahybridization probe comprising the polynucleotide of claim 6 and adetectable label.
 9. An isolated polynucleotide encoding the amino acidsequence of SEQ ID NO:2.
 10. An isolated polynucleotide of claim 9,wherein said polynucleotide comprises SEQ ID NO.4.
 11. A hybridizationprobe comprising a polynucleotide of claim 9 and a detectable label. 12.A hybridization probe comprising the polynucleotide of claim 10 and adetectable label.
 13. A polynucleotide which is fully complementary tothe polynucleotide of claim
 9. 14. A polynucleotide which is fullycomplementary to the polynucleotide of claim
 10. 15. A hybridizationprobe comprising the polynucleotide of claim 13 and a detectable label.16. A hybridization probe comprising the polynucleotide of claim 14 anda detectable label.